THE INTERFERON-STIMULATED GENE 54 K PROMOTER CONTAINS TWO ADJACENT FUNCTIONAL INTERFERON-STIMULATED RESPONSE ELEMENTS OF DIFFERENT STRENGTH, WHICH ACT SYNERGISTICALLY FOR MAXIMAL INTERFERON-ALPHA INDUCIBILITY

摘要: The interferon-alpha(IFN-alpha)-regulated hamster ISG-54 K gene, which is activated in CHO-12 cells at least 40-fold, was isolated and the promoter region characterized detail. Sequence analysis revealed presence of two elements, closely related to interferon-stimulated-response-element (ISRE) consensus sequence [AGTTTCNNTTTC(CT)]. putative ISRE-I (GGTTTCAATTTCT) located position -97 -85; ISRE-II (AGTTTTACTTTCT), differs three positions from ISRE-I, found directly upstream -110 -98. In a transient transfection assay wild-type ISG-54K-promoter-chloramphenicol-acetyl-transferase (CAT) reporter construct showed 40-80-fold induction, offering an excellent model study functional properties ISRE. To find out whether both elements were interferon regulation promoter, selected point mutations introduced -85 flanking sequences. (mutated) linked CAT gene transiently expressed CHO absence murine (Mu)IFN-alpha 6. Transfections that (ISRE-I) -98 (ISRE-II) segment needed for optimal induction promoter. However, has approximately sevenfold stronger activity compared ISRE-II. Sequential substitution bases, differ T -105 causes lower Transfection constructs, replaced by ISRE-II, generates with segments, vice versa (two ISRE-I), provided further evidence role IFN-alpha induction. Importantly, all data obtained studies show ISRE cooperate synergistically. mechanism synergism most probably indirect interaction between transcription factors binding ISRE, because increase spacial arrangement complete helical turn or half did not result substantial decrease activity.